Polymerase inhibitors and related compositions and methods

ABSTRACT

The present disclosure includes compositions and methods for improved DNA amplification reactions. In particular, the present disclosure provides compositions and methods for hot-start PCR applications using DNA polymerase inhibitors that minimize non-specific DNA amplification by inactivating DNA polymerase at lower temperatures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/778,590, filed on Dec. 12, 2018, the entire contents of which arefully incorporated herein by reference.

FIELD

Provided herein are compositions and methods for improved DNAamplification reactions. In particular, the present disclosure providescompositions and methods for hot-start PCR applications using DNApolymerase inhibitors that minimize non-specific DNA amplification byinactivating DNA polymerase at lower temperatures.

BACKGROUND

Biological PCR is a rapid and simple method for specifically amplifyinga target DNA sequence in an exponential manner (e.g., Saiki, et al.,Science 239:487-4391 (1988); herein incorporated by reference in itsentirety). Polymerase chain reaction (PCR) is a technology in molecularbiology used to amplify a single copy or a few copies of a piece of DNAacross several orders of magnitude, generating thousands to millions ofcopies of a particular DNA sequence. Hot-start PCR is a modified form ofpolymerase chain reaction (PCR) that avoids a non-specific amplificationof DNA by inactivating the DNA polymerase at lower temperatures.Initially, hot-start PCR was performed by withholding the Mg²⁺, dNTP, orenzyme until immediately before initial denaturation during cyclingbegins. Alternatively, hot-start PCR can be achieved by separating thereaction components with a wax bead barrier that melts as the mixture isheated during the initial denaturation step of the PCR.

Other hot-start PCR methodologies include the use of specific antibodiesor reversible chemical modifications of the polymerase (e.g.,modifications of the lysine with organic acid anhydride) to block theactivity of the DNA polymerase at lower temperature. An initialactivation step at 95° C. is therefore required for activation of thepolymerase. This step will both denature antibodies linked to the activecenter of the enzyme and also remove any lysine modifications, made withacid anhydride, from the chemically modified DNA polymerase. Forexample, anti-Taq antibodies reduce Taq polymerase activity below 72°C., the optimal temperature at which the enzyme extends the primers.When the specific antibodies detach from Taq-polymerase, theamplification proceeds with greater specificity.

However, there are significant disadvantages of using antibody-basedhot-start PCR or hot-start PCR based on chemical modifications of theDNA polymerase. For example, antibody-based hot-start PCR requires theuse of a different antibody for each enzyme used in a PCR reaction, andchemical modifications to the DNA polymerase can require longer times at95° C. to activate the enzymes and not all enzyme activity may berecovered.

SUMMARY

Provided herein are compounds of formula (I):

-   -   or a salt thereof,    -   wherein:    -   A is selected from aryl, heteroaryl, and heterocyclyl, each of        which may be optionally substituted with 1, 2, or 3        substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH; and    -   n is 1 or 2.

In some embodiments, A is phenyl that is unsubstituted or substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, and halo. In some embodiments, A is a 5- or 6-memberedmonocyclic heteroaryl that is unsubstituted or substituted with 1, 2, or3 substituents independently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy,and halo. In some embodiments, A is a bicyclic heterocyclyl group thatis unsubstituted. In some embodiments, A is selected from2,3-dihydrobenzofuranyl and chromanyl.

In some embodiments, R¹ is C₈-C₁₄ alkyl. In some embodiments, R¹ is C₁₂alkyl.

In some embodiments, R² is hydrogen. In some embodiments, R² is —COOH.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, the compound is in the form of an alkali metalsalt.

In some embodiments, the compound is selected from:

Provided herein are compositions comprising a compound of formula (I) asdescribed herein, and a DNA polymerase. In some embodiments, the DNApolymerase is a thermostable DNA polymerase. In some embodiments, thethermostable DNA polymerase is selected from the group consisting of:Taq, Tca, Tfu, Tbr, Tth, Tih, Tfi, Tli, Tfl, Pfu, Pwo, KOD, Tma, Tne,Bst, Pho, Sac, Sso, ES4, or a mutant, variant, or derivative thereof. Insome embodiments, the compound is bound to the DNA polymerase. In someembodiments, the compound inhibits the activity of the DNA polymerase.

In some embodiments, the composition comprises one or more nucleic acidamplification reagents. In some embodiments, the one or moreamplification reagents are selected from the group consisting of:deoxynucleotide triphosphates, buffer, a magnesium salt (e.g., MgCl₂ orMgSO₄), an oligonucleotide primer, and a nucleic acid template.

Provided herein are compositions comprising a compound of formula (I) asdescribed herein, and one or more nucleic acid amplification reagents.In some embodiments, the one or more amplification reagents are selectedfrom the group consisting of: a polymerase, deoxynucleotidetriphosphates, buffer, a magnesium salt (e.g., MgCl₂ or MgSO₄), anoligonucleotide primer, and a nucleic acid template.

Provided herein are compositions comprising a compound of formula (II):

-   -   or a salt thereof,    -   wherein:    -   A is selected from a monocyclic or bicyclic aryl, heteroaryl, or        heterocyclyl group, each of which may be optionally substituted        with 1, 2, or 3 substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH;    -   n is 1 or 2; and    -   R³ is selected from —COOH and —SO₃X, wherein X is selected from        hydrogen, an alkali metal cation, and an ammonium cation; and    -   one or more nucleic acid amplification reagents.

In some embodiments, A is phenyl that is unsubstituted or substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, and halo. In some embodiments, A is a 5- or 6-memberedmonocyclic heteroaryl that is unsubstituted or substituted with 1, 2, or3 substituents independently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy,and halo. In some embodiments, A is a bicyclic heterocyclyl group thatis unsubstituted. In some embodiments, A is selected from2,3-dihydrobenzofuranyl and chromanyl.

In some embodiments, R¹ is C₈-C₁₄ alkyl. In some embodiments, R₁ is C₁₂alkyl.

In some embodiments, R² is hydrogen. In some embodiments, R² is —COOH.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, R³ is —COOH. In some embodiments, R³ is —SO₃X, andX is a sodium cation.

In some embodiments, the compound is in the form of an alkali metalsalt.

In some embodiments, compound of formula (II) is selected from:

In some embodiments, the one or more amplification reagents are selectedfrom the group consisting of: a polymerase, deoxynucleotidetriphosphates, buffer, a magnesium salt (e.g., MgCl₂ or MgSO₄), anoligonucleotide primer, and a nucleic acid template.

Provided herein are methods of temperature-dependent inhibition ofpolymerase activity comprising contacting a DNA polymerase with acompound of formula (II):

-   -   or a salt thereof,    -   wherein:    -   A is selected from a monocyclic or bicyclic aryl, heteroaryl, or        heterocyclyl group, each of which may be optionally substituted        with 1, 2, or 3 substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH;    -   n is 1 or 2; and    -   R³ is selected from —COOH and —SO₃X, wherein X is selected from        hydrogen, an alkali metal cation, and an ammonium cation.

In some embodiments, A is phenyl that is unsubstituted or substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, and halo. In some embodiments, A is a 5- or 6-memberedmonocyclic heteroaryl that is unsubstituted or substituted with 1, 2, or3 substituents independently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy,and halo. In some embodiments, A is a bicyclic heterocyclyl group thatis unsubstituted. In some embodiments, A is selected from2,3-dihydrobenzofuranyl and chromanyl.

In some embodiments, R¹ is C₈-C₁₄ alkyl. In some embodiments, R₁ is C₁₂alkyl.

In some embodiments, R² is hydrogen. In some embodiments, R² is —COOH.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, R³ is —COOH. In some embodiments, R³ is —SO₃X,wherein X is a sodium cation.

In some embodiments, the compound of formula (II) is selected from:

In some embodiments, the polymerase is a thermostable DNA polymerase. Insome embodiments, the thermostable DNA polymerase is selected from thegroup consisting of: Taq, Tca, Tfu, Tbr, Tth, Tih, Tfi, Tli, Tfl, Pfu,Pwo, KOD, Tma, Tne, Bst, Pho, Sac, Sso, ES4, or a mutant, variant, orderivative thereof.

Provided herein are methods of activating an inhibited polymerasecomprising exposing a DNA polymerase inhibited by a compound of formula(II):

or a salt thereof,

-   -   wherein:    -   A is selected from a monocyclic or bicyclic aryl, heteroaryl, or        heterocyclyl group, each of which may be optionally substituted        with 1, 2, or 3 substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH;    -   n is 1 or 2; and    -   R³ is selected from —COOH and —SO₃X, wherein X is selected from        hydrogen, an alkali metal cation, and an ammonium cation; and    -   heating to a temperature above 80° C. to activate the DNA        polymerase.

In some embodiments, the compound is a compound of formula (I):

-   -   or a salt thereof,    -   wherein:    -   A is selected from aryl, heteroaryl, and heterocyclyl, each of        which may be optionally substituted with 1, 2, or 3        substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH; and    -   n is 1 or 2.

In some embodiments, the polymerase is a thermostable DNA polymerase. Insome embodiments, the thermostable DNA polymerase is selected from thegroup consisting of: Taq, Tca, Tfu, Tbr, Tth, Tih, Tfi, Tli, Tfl, Pfu,Pwo, KOD, Tma, Tne, Bst, Pho, Sac, Sso, ES4, or a mutant, variant, orderivative thereof. In some embodiments, the temperature is above 90° C.

Provided herein are methods of amplifying a nucleic acid comprising:

-   -   (a) adding amplification reagents and a nucleic acid template to        a DNA polymerase inhibited by a compound of formula (II):

-   -   or a salt thereof,    -   wherein:    -   A is selected from a monocyclic or bicyclic aryl, heteroaryl, or        heterocyclyl group, each of which may be optionally substituted        with 1, 2, or 3 substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH;    -   n is 1 or 2; and    -   R³ is selected from —COOH and —SO₃X, wherein X is selected from        hydrogen, an alkali metal cation, and an ammonium cation;    -   (b) heating to a temperature of at least 80° C. to activate the        DNA polymerase; and    -   (c) running through a thermal cycling protocol of appropriate        times and temperatures for the amplification reagents and a        nucleic acid template.

In some embodiments, the amplification reagents comprise:deoxynucleotide triphosphates, buffer, a magnesium salt (e.g., MgCl₂ orMgSO₄), and an oligonucleotide primer. In some embodiments, theamplification reagents comprise forward and reverse primers for a targeton the nucleic acid template. In some embodiments, the DNA polymerase isactivated by heating to a temperature above 80° C. In some embodiments,the thermal cycling protocol comprises a three temperature cycle of (i)a high temperature denaturation step, (ii) a low temperature annealingstep, and (iii) a middle temperature extension step, repeated 5 or moretimes in succession. In some embodiments, the denaturation, annealing,and extension steps are repeated 20 or more times in succession. In someembodiments, the thermal cycling protocol comprises a two temperaturecycle of a high temperature denaturation step, a middle/low temperatureannealing/extension step repeated 5 or more times in succession. In someembodiments, the denaturation and annealing/extension steps are repeated20 or more times in succession.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B include representative images of agarose gels used toevaluate the amplification products of various hot-start PCR reactionsperformed with and without two different polymerase inhibitors (compound#7124 in FIG. 1A; compound #7261 in FIG. 1B).

FIG. 2 includes a representative table of the results of varioushot-start PCR reactions performed with different polymerase inhibitors(i.e., compound #7437, compound #7438, and compound #7439; variations ofcompound #7261).

FIG. 3 includes a representative table of the results of varioushot-start PCR reactions performed with different polymerase inhibitors(i.e., compound #7489 and compound #7490; variations of compound #7124).

FIG. 4 includes a representative table of the results of varioushot-start PCR reactions performed with different polymerase inhibitors(i.e., compound #7487 and compound #7488; variations of compound #7124).

FIG. 5 includes a representative table of the results of varioushot-start PCR reactions performed with different polymerase inhibitors(i.e., compound #7491 and compound #7493; variations of compound #7124).

FIG. 6 includes a representative table of the results of varioushot-start PCR reactions performed with different polymerase inhibitors(i.e., compound #7486 and compound #7495; variations of compound #7124).

FIG. 7 includes a representative graph of the results of Taq DNAPolymerase activity at 22° C. for a titration of a polymerase inhibitor(i.e. compound #7124), which can be used to estimate the IC₅₀.

FIG. 8 includes a representative table of results of IC₅₀ values fordifferent polymerase inhibitors (i.e. compound #7124, compound #7126,compound #7127, compound #7123, compound #7125, compound #6966, andSDS).

FIG. 9 includes a representative table of the results of varioushot-start PCR reactions performed with polymerase inhibitor compound#7124 and polymerase enzyme at the various concentrations shown.

FIG. 10 includes a representative table of the results of varioushot-start PCR reactions performed with polymerase inhibitor compound#7261 and polymerase enzyme at the various concentrations shown.

FIG. 11 includes a representative image of an agarose gel used toevaluate the CCR5 gene amplification products of various hot-start PCRreactions performed with and without two different polymerase inhibitors(compound #7124 and compound #7261) using human genomic DNA as atemplate.

FIG. 12 includes a representative image of an agarose gel used toevaluate the Human Dystrophin Alu 7-2 gene amplification products ofvarious hot-start PCR reactions performed with and without two differentpolymerase inhibitors (compound #7124 and compound #7261) using humangenomic DNA as a template.

DETAILED DESCRIPTION

Provided herein are compositions and methods for improved DNAamplification reactions. In particular, the present disclosure providescompositions and methods for hot-start PCR applications using DNApolymerase inhibitors that minimize non-specific DNA amplification byinactivating DNA polymerase at lower temperatures.

Embodiments of the present disclosure provide novel thermally-labilesmall molecule polymerase inhibitors for PCR applications (e.g.,hot-start PCR). The addition of thermally-labile molecules toamplification reactions provides improved hot-start PCR applications byinhibiting DNA polymerase activity at lower temperatures. Unlikeantibody-based hot-start PCR, use of a thermally-labile small moleculepolymerase inhibitor is a simple and universal approach widelyapplicable to different DNA polymerases that may be used in hot-startPCR applications. Embodiments provided herein also eliminate undesirednuclease activity associated with DNA polymerases and avoid non-specificproduct amplification at low temperatures, thus producing an amplifiedproduct with higher specificity and yield.

As would be recognized by one of ordinary skill in the art based on thepresent disclosure, many challenging PCR reactions and analyses benefitfrom a hot start, and in some cases, a hot start is required to amplifya desired DNA target. This includes reactions which have very low copynumbers of target (e.g., 1 HIV genome per 10,000 cells), denatured DNA(e.g., many DNA extraction procedures include a boiling step so that thetemplate is single-stranded during reaction setup), or contaminated DNA(e.g., DNA from soil or feces and/or DNA containing large amounts ofRNA). Additionally, other challenges that can be addressed withhot-start PCR include poorly designed primers, multiplexingamplifications into single reactions, and having similar (but notidentical) target sites in the DNA template. The novel thermally-labilesmall molecule DNA polymerase inhibitors of the present disclosureaddress these challenges.

Section headings as used in this section and the entire disclosureherein are merely for organizational purposes and are not intended to belimiting.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentdisclosure. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Sorrell, Organic Chemistry, 2^(nd) edition, UniversityScience Books, Sausalito, 2006; Smith, March's Advanced OrganicChemistry: Reactions, Mechanism, and Structure, 7^(th) Edition, JohnWiley & Sons, Inc., New York, 2013; Larock, Comprehensive OrganicTransformations, 3^(rd) Edition, John Wiley & Sons, Inc., New York,2018; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987; the entirecontents of each of which are incorporated herein by reference.

The term “alkoxy”, as used herein, refers to an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.

The term “alkyl”, as used herein, means a straight or branched saturatedhydrocarbon chain containing from 1 to 30 carbon atoms, for example 1 to16 carbon atoms (C₁-C₁₆ alkyl), 1 to 14 carbon atoms (C₁-C₁₄ alkyl), 1to 12 carbon atoms (C₁-C₁₂ alkyl), 1 to 10 carbon atoms (C₁-C₁₀ alkyl),1 to 8 carbon atoms (C₁-C₈ alkyl), 1 to 6 carbon atoms (C₁-C₆ alkyl), 1to 4 carbon atoms (C₁-C₄ alkyl), 6 to 20 carbon atoms (C₆-C₂₀ alkyl), or8 to 14 carbon atoms (C₈-C₁₄ alkyl). Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, andn-dodecyl.

The term “alkenyl”, as used herein, refers to a straight or branchedhydrocarbon chain containing from 2 to 30 carbon atoms and containing atleast one carbon-carbon double bond. Representative examples of alkenylinclude, but are not limited to, ethenyl, 2-propenyl,2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl,2-methyl-1-heptenyl, and 3-decenyl.

The term “alkynyl”, as used herein, refers to a straight or branchedhydrocarbon chain containing from 2 to 30 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited to, ethynyl, propynyl, and butynyl.

The term “aryl”, as used herein, refers to a phenyl group, or a bicyclicor tricyclic aromatic fused ring system. Bicyclic fused ring systems areexemplified by a phenyl group appended to the parent molecular moietyand fused to a phenyl group. Tricyclic fused ring systems areexemplified by a phenyl group appended to the parent molecular moietyand fused to two other phenyl groups. Representative examples ofbicyclic aryls include, but are not limited to, naphthyl. Representativeexamples of tricyclic aryls include, but are not limited to,anthracenyl.

The term “cycloalkyl”, as used herein, refers to a saturated carbocyclicring system containing three to ten carbon atoms and zero heteroatoms.The cycloalkyl may be monocyclic, bicyclic, bridged, fused, orspirocyclic. Representative examples of cycloalkyl include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl,bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, andbicyclo[5.2.0]nonanyl.

The term “cycloalkenyl”, as used herein, means a non-aromatic monocyclicor multicyclic ring system containing at least one carbon-carbon doublebond and preferably having from 5-10 carbon atoms per ring. Exemplarymonocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl orcycloheptenyl.

The term “halogen” or “halo”, as used herein, means F, Cl, Br, or I.

The term “haloalkyl”, as used herein, means an alkyl group, as definedherein, in which one, two, three, four, five, six, seven or eighthydrogen atoms are replaced by a halogen.

The term “heteroaryl”, as used herein, refers to an aromatic monocyclicring or an aromatic bicyclic ring system or an aromatic tricyclic ringsystem. The aromatic monocyclic rings are five- or six-membered ringscontaining at least one heteroatom independently selected from the groupconsisting of N, O, and S (e.g. 1, 2, 3, or 4 heteroatoms independentlyselected from 0, S, and N). The five-membered aromatic monocyclic ringshave two double bonds, and the six-membered aromatic monocyclic ringshave three double bonds. The bicyclic heteroaryl groups are exemplifiedby a monocyclic heteroaryl ring appended fused to a monocyclic arylgroup, as defined herein, or a monocyclic, heteroaryl group, as definedherein. The tricyclic heteroaryl groups are exemplified by a monocyclicheteroaryl ring fused to two rings independently selected from amonocyclic aryl group, as defined herein or a monocyclic heteroarylgroup as defined herein. Representative examples of monocyclicheteroaryl include, but are not limited to, pyridinyl (includingpyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl,pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl,1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl,furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl.Representative examples of bicyclic heteroaryl include, but are notlimited to, benzimidazolyl, benzodioxolyl, benzofuranyl,benzooxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl,benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl,imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl,isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl,quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl,thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl. Representativeexamples of tricyclic heteroaryl include, but are not limited to,dibenzofuranyl and dibenzothienyl. The monocyclic, bicyclic, andtricyclic heteroaryls are connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within the rings.

The term “heterocycle” or “heterocyclic”, as used herein, means amonocyclic heterocycle, a bicyclic heterocycle, or a tricyclicheterocycle. The monocyclic heterocycle is a three-, four-, five-, six-,seven-, or eight-membered ring containing at least one heteroatomindependently selected from the group consisting of O, N, and S. Thethree- or four-membered ring contains zero or one double bond, and oneheteroatom selected from the group consisting of O, N, and S. Thefive-membered ring contains zero or one double bond and one, two orthree heteroatoms selected from the group consisting of O, N and S. Thesix-membered ring contains zero, one, or two double bonds and one, two,or three heteroatoms selected from the group consisting of O, N, and S.The seven- and eight-membered rings contains zero, one, two, or threedouble bonds and one, two, or three heteroatoms selected from the groupconsisting of O, N, and S. Representative examples of monocyclicheterocycles include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl,piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl,pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl,thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl,thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclicheterocycle is a monocyclic heterocycle fused to a phenyl group, or amonocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclicheterocycle fused to a monocyclic cycloalkenyl, or a monocyclicheterocycle fused to a monocyclic heterocycle, or a spiro heterocyclegroup, or a bridged monocyclic heterocycle ring system in which twonon-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2,3, or 4 carbon atoms, or an alkenylene bridge of two, three, or fourcarbon atoms. Representative examples of bicyclic heterocycles include,but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl,2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl,2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl,azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl),2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl,octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclicheterocycles are exemplified by a bicyclic heterocycle fused to a phenylgroup, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or abicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclicheterocycle fused to a monocyclic heterocycle, or a bicyclic heterocyclein which two non-adjacent atoms of the bicyclic ring are linked by analkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridgeof two, three, or four carbon atoms. Examples of tricyclic heterocyclesinclude, but are not limited to, octahydro-2,5-epoxypentalene,hexahydro-2H-2,5-methanocyclopenta[b]furan,hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane(1-azatricyclo[3.3.1.1^(3,7)]decane), and oxa-adamantane(2-oxatricyclo[3.3.1.1^(3,7)]decane). The monocyclic, bicyclic, andtricyclic heterocycles are connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within the rings.

The term “hydroxy”, as used herein, means an —OH group.

In some instances, the number of carbon atoms in a group (e.g., alkyl,alkoxy, or cycloalkyl) is indicated by the prefix “C_(x)-C_(y)-”,wherein x is the minimum and y is the maximum number of carbon atoms inthe group. Thus, for example, “C₁-C₃-alkyl” refers to an alkyl groupcontaining from 1 to 3 carbon atoms.

The term “substituent” refers to a group substituted on an atom of theindicated group.

When a group or moiety can be substituted, the term “substituted”indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in someembodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens onthe group indicated in the expression using “substituted” can bereplaced with a selection of recited indicated groups or with a suitablegroup known to those of skill in the art (e.g., one or more of thegroups recited below). Substituent groups include, but are not limitedto, halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl,fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy,heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle,cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl,alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino,alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino,sulfinylamino, sulfonyl, alkyl sulfonyl, aryl sulfonyl, aminosulfonyl,sulfinyl, —COOH, ketone, amide, carbamate, and acyl.

For compounds described herein, groups and substituents thereof may beselected in accordance with permitted valence of the atoms and thesubstituents, such that the selections and substitutions result in astable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”,“contain(s)”, and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a”, “and”, and “the” include plural references unless the contextclearly dictates otherwise. Many embodiments herein are described usingopen “comprising” language. Such embodiments encompass multiple closed“consisting of” and/or “consisting essentially of” embodiments, whichmay alternatively be claimed or described using such language. Thepresent disclosure also contemplates other embodiments “comprising”,“consisting of”, and “consisting essentially of” the embodiments orelements presented herein, whether explicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Isolated polynucleotide” as used herein may mean a polynucleotide(e.g., of genomic, cDNA, or synthetic origin, or a combination thereof)that, by virtue of its origin, the isolated polynucleotide is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

“Peptide” and “polypeptide”, as used herein, and unless otherwisespecified, refer to polymer compounds of two or more amino acids joinedthrough the main chain by peptide amide bonds (—C(O)NH—). The term“peptide” typically refers to short amino acid polymers (e.g., chainshaving fewer than 25 amino acids), whereas the term “polypeptide”typically refers to longer amino acid polymers (e.g., chains having morethan 25 amino acids).

“Sample”, “test sample”, “specimen”, “sample from a subject”, and“patient sample” as used herein may be used interchangeable and may be asample of blood, such as whole blood, tissue, urine, serum, plasma,amniotic fluid, cerebrospinal fluid, placental cells or tissue,endothelial cells, leukocytes, or monocytes. The sample can be useddirectly as obtained from a patient or can be pre-treated, such as byfiltration, distillation, extraction, concentration, centrifugation,inactivation of interfering components, addition of reagents, and thelike, to modify the character of the sample in some manner as discussedherein or otherwise as is known in the art.

“Sequence identity” refers to the degree two polymer sequences (e.g.,peptide, polypeptide, nucleic acid, etc.) have the same sequentialcomposition of monomer subunits. The term “sequence similarity” refersto the degree with which two polymer sequences (e.g., peptide,polypeptide, nucleic acid, etc.) have similar polymer sequences. Forexample, similar amino acids are those that share the same biophysicalcharacteristics and can be grouped into the families, e.g., acidic(e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine),non-polar (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), and uncharged polar (e.g.,glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine).The “percent sequence identity” (or “percent sequence similarity”) iscalculated by: (1) comparing two optimally aligned sequences over awindow of comparison (e.g., the length of the longer sequence, thelength of the shorter sequence, a specified window), (2) determining thenumber of positions containing identical (or similar) monomers (e.g.,same amino acids occurs in both sequences, similar amino acid occurs inboth sequences) to yield the number of matched positions, (3) dividingthe number of matched positions by the total number of positions in thecomparison window (e.g., the length of the longer sequence, the lengthof the shorter sequence, a specified window), and (4) multiplying theresult by 100 to yield the percent sequence identity or percent sequencesimilarity. For example, if peptides A and B are both 20 amino acids inlength and have identical amino acids at all but 1 position, thenpeptide A and peptide B have 95% sequence identity. If the amino acidsat the non-identical position shared the same biophysicalcharacteristics (e.g., both were acidic), then peptide A and peptide Bwould have 100% sequence similarity. As another example, if peptide C is20 amino acids in length and peptide D is 15 amino acids in length, and14 out of 15 amino acids in peptide D are identical to those of aportion of peptide C, then peptides C and D have 70% sequence identity,but peptide D has 93.3% sequence identity to an optimal comparisonwindow of peptide C. For the purpose of calculating “percent sequenceidentity” (or “percent sequence similarity”) herein, any gaps in alignedsequences are treated as mismatches at that position.

“Subsequence” refers to peptide or polypeptide that has 100% sequenceidentify with another, larger peptide or polypeptide. The subsequence isa perfect sequence match for a portion of the larger amino acid chain.

“Substantially”, as used herein, means that the recited characteristic,parameter, and/or value need not be achieved exactly, but thatdeviations or variations, including for example, tolerances, measurementerror, measurement accuracy limitations, and other factors known toskill in the art, may occur in amounts that do not preclude the effectthe characteristic was intended to provide. A characteristic or featurethat is substantially absent be one that is within the noise, beneathbackground, below the detection capabilities of the assay being used, ora small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%,<0.000001%, <0.0000001%) of the significant characteristic.

“Variant” is used herein to describe a peptide or polypeptide thatdiffers in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. “SNP” refers to a variant that is a singlenucleotide polymorphism. Representative examples of “biologicalactivity” include the ability to be bound by a specific antibody or topromote an immune response. Variant is also used herein to describe aprotein with an amino acid sequence that is substantially identical to areferenced protein with an amino acid sequence that retains at least onebiological activity. A conservative substitution of an amino acid (e.g.,replacing an amino acid with a different amino acid of similarproperties, such as hydrophilicity, degree, and distribution of chargedregions) is recognized in the art as typically involving a minor change.These minor changes can be identified, in part, by considering thehydropathic index of amino acids, as understood in the art. Thehydropathic index of an amino acid is based on a consideration of itshydrophobicity and charge. It is known in the art that amino acids ofsimilar hydropathic indexes can be substituted and still retain proteinfunction. In one aspect, amino acids having hydropathic indexes of ±2are substituted. The hydrophilicity of amino acids can also be used toreveal substitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a peptide permits calculation of the greatest local averagehydrophilicity of that peptide, a useful measure that has been reportedto correlate well with antigenicity and immunogenicity. Substitution ofamino acids having similar hydrophilicity values can result in peptidesretaining biological activity, for example immunogenicity, as isunderstood in the art. Substitutions may be performed with amino acidshaving hydrophilicity values within ±2 of each other. Both thehydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. For example,any nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those that are well known and commonly used in the art. Themeaning and scope of the terms should be clear; in the event, however ofany latent ambiguity, definitions provided herein take precedent overany dictionary or extrinsic definition. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

2. DNA POLYMERASE INHIBITORS

The present disclosure includes materials and methods related tohot-start PCR using novel thermally-labile small molecule DNA polymeraseinhibitors. In accordance with these embodiments, the present disclosureincludes DNA polymerase inhibitors comprising a carbon chain tail (R¹),a core group (A) that allows for breakdown of the molecule, and a headgroup.

In some embodiments, the disclosure provides a compound of formula (I):

-   -   or a salt thereof,    -   wherein:    -   A is selected from aryl, heteroaryl, and heterocyclyl, each of        which may be optionally substituted with 1, 2, or 3        substituents;    -   R¹ is C₆-C₂₀ alkyl;    -   R² is selected from hydrogen and —COOH; and    -   n is 1 or 2.

In some embodiments, A is phenyl, which is unsubstituted or substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, and halo. In some embodiments, A is phenyl that issubstituted with 1, 2, 3, or 4 substituents independently selected frommethyl, methoxy, and isopropoxy. In some embodiments, A is phenylsubstituted with 1 substituent selected from methoxy and isopropoxy. Insome embodiments, A is phenyl substituted with 2 substituentsindependently selected from methyl and methoxy. In some embodiments, Ais phenyl substituted with 3 substituents independently selected frommethyl and methoxy. In some embodiments, A is phenyl substituted with 4substituents independently selected from methyl and methoxy.

In some embodiments, A is a 5- or 6-membered monocyclic heteroaryl thatis unsubstituted or substituted with 1, 2, or 3 substituentsindependently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, and halo. In someembodiments, A is a 5-membered monocyclic heteroaryl having 1, 2, or 3heteroatoms independently selected from O, N, and S, and which isunsubstituted or substituted with 1, 2, or 3 substituents independentlyselected from C₁-C₄ alkyl, C₁-C₄ alkoxy, and halo. In some embodiments,A is a 5-membered monocyclic heteroaryl having one heteroatom selectedfrom O, N, and S, and which is unsubstituted or substituted with 1 or 2substituents independently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, andhalo. In some embodiments, A is furanyl that is substituted with 1substituent selected from halo (e.g., chloro or bromo).

In some embodiments, A is a bicyclic heterocyclyl group that isunsubstituted or substituted with 1, 2, or 3 substituents independentlyselected from C₁-C₄ alkyl, C₁-C₄ alkoxy, and halo. In some embodiments,A is a bicyclic heterocyclyl group that is unsubstituted. In someembodiments, A is selected from 2,3-dihydrobenzofuranyl and chromanyl.

In some embodiments, R¹ is C₈-C₁₄ alkyl. In some embodiments, R¹ is C₁₂alkyl.

In some embodiments, R² is hydrogen. In some embodiments, R² is —COOH.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, the compound is in the from of an alkali metal saltsuch as a sodium salt.

In some embodiments, the compound of formula (I) is selected from:

In another aspect, the disclosure provides compositions and methods,such as those further described herein, that use a compound of formula(II):

-   -   or a salt thereof,    -   wherein:    -   A is selected from a monocyclic or bicyclic aryl, heteroaryl, or        heterocyclyl group, each of which may be optionally substituted        with 1, 2, or 3 substituents;    -   R¹ is C₆-C₁₀ alkyl;    -   R² is selected from hydrogen and —COOH;    -   n is 1 or 2; and    -   R³ is selected from —COOH and —SO₃X,    -   wherein X is selected from hydrogen, an alkali metal cation, or        an ammonium cation.

In some embodiments, A is phenyl, which is unsubstituted or substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, and halo. In some embodiments, A is phenyl that issubstituted with 1, 2, 3, or 4 substituents independently selected frommethyl, methoxy, and isopropoxy. In some embodiments, A is phenylsubstituted with 1 substituent selected from methoxy and isopropoxy. Insome embodiments, A is phenyl substituted with 2 substituentsindependently selected from methyl and methoxy. In some embodiments, Ais phenyl substituted with 3 substituents independently selected frommethyl and methoxy. In some embodiments, A is phenyl substituted with 3substituents independently selected from methyl and methoxy.

In some embodiments, A is a 5- or 6-membered monocyclic heteroaryl thatis unsubstituted or substituted with 1, 2, or 3 substituentsindependently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, and halo. In someembodiments, A is a 5-membered monocyclic heteroaryl having 1, 2, or 3heteroatoms independently selected from O, N, and S, and which isunsubstituted or substituted with 1, 2, or 3 substituents independentlyselected from C₁-C₄ alkyl, C₁-C₄ alkoxy, and halo. In some embodiments,A is a 5-membered monocyclic heteroaryl having one heteroatom selectedfrom O, N, and S, and which is unsubstituted or substituted with 1 or 2substituents independently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, andhalo. In some embodiments, A is furanyl that is substituted with 1substituent selected from halo (e.g., chloro or bromo).

In some embodiments, A is a bicyclic heterocyclyl group that isunsubstituted or substituted with 1, 2, or 3 substituents independentlyselected from C₁-C₄ alkyl, C₁-C₄ alkoxy, and halo. In some embodiments,A is a bicyclic heterocyclyl group that is unsubstituted. In someembodiments, A is selected from 2,3-dihydrobenzofuranyl and chromanyl.

In some embodiments, R¹ is C₈-C₁₄ alkyl. In some embodiments, R¹ is C₁₂alkyl.

In some embodiments, R² is hydrogen. In some embodiments, R² is —COOH.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, R³ is —COOH. In some embodiments, R³ is —SO₃X,wherein X is a sodium cation.

In some embodiments, n is 2 and R³ is —SO₃X, wherein X is Na.

In some embodiments, R¹ is C₁₂ alkyl, R² is hydrogen, n is 2, and R³ is—SO₃X, wherein X is Na.

In some embodiments, the compound of formula (II) is selected from:

Compounds of formula (I) and formula (II) can be in the form of a salt.A neutral form of the compound may be regenerated by contacting the saltwith a base or acid and isolating the parent compound in a conventionalmanner. The parent form of the compound differs from the various saltforms in certain physical properties, such as solubility in polarsolvents, but otherwise the salts are equivalent to the parent form ofthe compound for the purposes of this disclosure.

In particular, compounds of formula (I) include at least one —COOHmoiety that may be anionic (i.e. —COO⁻) and form a salt with a suitablecation. Compounds of formula (II) include at least one —COOH moiety orat least one —SO₃H moiety that may be anionic (i.e. —COO⁻ or —SO₃ ⁻respectively) and form a salt with a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalcations such as Li⁺, Na⁺, and K⁺, alkaline earth cations such as Ca²⁺and Mg²⁺, and other cations. Sodium salts may be particularly suitable.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R₁ ⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺. Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids such as lysine and arginine.

If the compound is cationic or has a functional group that may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic,lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic,oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic,propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric,toluenesulfonic, and valeric.

Compounds of formula (I) and (II) can be prepared by a variety ofmethods. For example, compounds can be prepared as illustrated in Scheme1.

As illustrated in scheme 1, an aldehyde compound A can be reacted with asuitable Grignard reagent (e.g., a compound R¹—MgX where X is a halogensuch as bromo) to generate compound B. Reaction of compound B withp-nitrophenyl chloroformate to generate the corresponding carbonatecompound, which can be reacted with an appropriate amine to form thecompound of formula (II). One skilled in the art will recognize thatcompounds of formula (I) can be prepared similarly using compounds inwhich R³ is —COOH.

The compounds and intermediates herein may be isolated and purified bymethods well-known to those skilled in the art of organic synthesis.Examples of conventional methods for isolating and purifying compoundscan include, but are not limited to, chromatography on solid supportssuch as silica gel, alumina, or silica derivatized with alkylsilanegroups, by recrystallization at high or low temperature with an optionalpretreatment with activated carbon, thin-layer chromatography,distillation at various pressures, sublimation under vacuum, andtrituration as described for instance in “Vogel's Textbook of PracticalOrganic Chemistry,” 5th edition (1989), by Furniss, Hannaford, Smith,and Tatchell, pub. Longman Scientific & Technical, Essex CM20 2JE,England.

Reaction conditions and reaction times for each individual step can varydepending on the particular reactants employed and substituents presentin the reactants used. Specific procedures are provided in the Examplessection. Reactions can be worked up in the conventional manner, e.g., byeliminating the solvent from the residue and further purified accordingto methodologies generally known in the art such as, but not limited to,crystallization, distillation, extraction, trituration, andchromatography. Unless otherwise described, the starting materials andreagents are either commercially available or can be prepared by oneskilled in the art from commercially available materials using methodsdescribed in the chemical literature. Starting materials, if notcommercially available, can be prepared by procedures selected fromstandard organic chemical techniques, techniques that are analogous tothe synthesis of known, structurally similar compounds, or techniquesthat are analogous to the above described schemes or the proceduresdescribed in the synthetic examples section.

Routine experimentations, including appropriate manipulation of thereaction conditions, reagents and sequence of the synthetic route,protection of any chemical functionality that cannot be compatible withthe reaction conditions, and deprotection at a suitable point in thereaction sequence of the method are included in the scope of theinvention. Suitable protecting groups, and the methods for protectingand deprotecting different substituents using such suitable protectinggroups, are well known to those skilled in the art; examples of whichcan be found in the treatise by PGM Wuts entitled “Greene's ProtectiveGroups in Organic Synthesis” (5th ed.), John Wiley & Sons, Inc. (2014),which is incorporated herein by reference in its entirety. Synthesis ofthe compounds of the invention can be accomplished by methods analogousto those described in the synthetic schemes described hereinabove and inspecific examples.

When an optically active form of a disclosed compound is required, itcan be obtained by carrying out one of the procedures described hereinusing an optically active starting material (prepared, for example, byasymmetric induction of a suitable reaction step) or by resolution of amixture of the stereoisomers of the compound or intermediates using astandard procedure (such as chromatographic separation,recrystallization or enzymatic resolution).

Similarly, when a pure geometric isomer of a compound is required, itcan be obtained by carrying out one of the above procedures using a puregeometric isomer as a starting material or by resolution of a mixture ofthe geometric isomers of the compound or intermediates using a standardprocedure such as chromatographic separation.

The synthetic schemes and specific examples as described areillustrative and are not to be read as limiting the scope of theinvention as it is defined in the claims. All alternatives,modifications, and equivalents of the synthetic methods and specificexamples are included within the scope of the claims.

3. PCR REAGENTS, COMPOSITIONS, AND KITS

Embodiments of the present disclosure include various reagents used tocarry out PCR reactions, including, but not limited to, hot-start PCRreactions. Such PCR reactions may be performed with any suitablereagents, as described herein, including the novel thermally-labilesmall molecule DNA polymerase inhibitors of the present disclosure. DNApolymerases that can be used in accordance with these embodimentsinclude, but are not limited to, any polymerase capable of replicating aDNA molecule. In some embodiments, DNA polymerases are thermostablepolymerases, which are especially useful in PCR applications.Thermostable polymerases are isolated from a wide variety ofthermophilic bacteria, such as Thermus aquaticus (Taq), Thermusbrockianus (Tbr), Thermus flavus (Tfl), Thermus ruber (Tru), Thermusthermophilus (Tth), Thermococcus litoralis (Tli) and other species ofthe Thermococcus genus, Thermoplasma acidophilum (Tac), Thermotoganeapolitana (Tne), Thermotoga maritima (Tma), and other species of theThermotoga genus, Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo) andother species of the Pyrococcus genus, Bacillus sterothermophilus (Bst),Sulfolobus acidocaldarius (Sac) Sulfolobus solfataricus (Sso),Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), andMethanobacterium thermoautotrophicum (Mth), and mutants, variants orderivatives thereof.

In some embodiments, DNA polymerases that can be used in accordance withthese embodiments include, but are not limited to, commerciallyavailable DNA polymerases (e.g., from Boehringer Mannheim Corp.,Indianapolis, Ind.; Life Technologies, Inc., Rockville, Md.; New EnglandBiolabs, Inc., Beverley, Mass.; Perkin Elmer Corp., Norwalk, Conn.;Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.; Qiagen, Inc.,Valencia, Calif.; Stratagene, La Jolla, Calif). In some embodiments, aDNA polymerase is Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi,Pfu, Pwo, KOD, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT®, DEEPVENT®,and active mutants, variants and derivatives thereof. Other DNApolymerases can be used in conjunction with the novel thermal labilesmall molecule DNA polymerase inhibitors provided herein, including DNApolymerases not specifically disclosed above, but would be known to oneof ordinary skill in the art based on the present disclosure.

In accordance with the embodiments provided herein, various other PCRreagents can include an amplification reagent, which may include atleast one primer or at least one pair of primers for amplification of anucleic acid target, at least one probe and/or dye to enable detectionof amplification, a ligase, a detergent (e.g., non-ionic detergents),nucleotides (dNTPs and/or NTPs), divalent magnesium ions, or anycombination thereof, among others that would be recognized by one ofordinary skill in the art based on the present disclosure. In someembodiments, an amplification reagent and/or a nucleic acid target eachmay be present at an effective amount, such as an amount sufficient toenable amplification of a desired nucleic acid target in the presence ofother necessary reagents, including, but not limited to, a thermostablereverse transcriptase and manganese.

In accordance with the embodiments provided herein, PCR reagents caninclude one or more primers, or any nucleic acid capable of, and/or usedfor, priming replication of a nucleic acid template. Generally, a primeris a shorter nucleic acid that is complementary to a longer template.During replication, the primer may be extended, based on the templatesequence, to produce a longer nucleic acid that is a complementary copyof the template. Extension may occur by successive addition ofindividual nucleotides (e.g., by the action of a polymerase) or byattachment of a block of nucleotides (e.g., by the action of a ligasejoining a pair of primers), among others. A primer may be DNA, RNA, ananalog thereof (e.g., an artificial nucleic acid), or any combinationthereof. A primer may have any suitable length, such as at least about10, 15, 20, or 30 nucleotides. Exemplary primers are synthesizedchemically. Primers may be supplied as at least one pair of primers foramplification of at least one nucleic acid target. A pair of primers maybe a sense primer and an antisense primer that collectively define theopposing ends (and thus the length) of a resulting amplicon.

In accordance with the embodiments provided herein, PCR reagents canalso include one or more probes, or any nucleic acid connected to atleast one label, such as at least one dye. A probe may be asequence-specific binding partner for a nucleic acid target and/oramplicon. The probe may be designed to enable detection of targetamplification based on fluorescence resonance energy transfer (FRET),including one or more nucleic acids connected to a pair of dyes thatcollectively exhibit fluorescence resonance energy transfer (FRET) whenproximate one another. The pair of dyes may provide first and secondemitters, or an emitter and a quencher, among others. Fluorescenceemission from the pair of dyes changes when the dyes are separated fromone another, such as by cleavage of the probe during primer extension(e.g., a 5′ nuclease assay, such as with a TAQMAN probe), or when theprobe hybridizes to an amplicon (e.g., a molecular beacon probe). Thenucleic acid portion of the probe may have any suitable structure ororigin, for example, the portion may be a locked nucleic acid, a memberof a universal probe library, or the like. In other cases, a probe andone of the primers of a primer pair may be combined in the samemolecule. For example, the primer-probe molecule may include a primersequence at its 3′ end and a molecular beacon-style probe at its 5′ end.With this arrangement, related primer-probe molecules labeled withdifferent dyes can be used in a multiplexed assay with the same reverseprimer to quantify target sequences differing by a single nucleotide(single nucleotide polymorphisms (SNPs)).

In some embodiments, PCR reagents can also include one or more labels orreporter molecules. A label includes any identifying and/ordistinguishing marker or identifier connected to or incorporated intoany entity such as a compound, biological particle (e.g., a DNA, a RNA,a cell, bacteria, spore, virus, or organelle), or droplet. A label may,for example, be a dye that renders an entity optically detectable and/oroptically distinguishable. Exemplary dyes used for labeling arefluorescent dyes (fluorophores) and fluorescence quenchers. A reporterincludes any compound or set of compounds that reports a condition suchas the extent of a reaction. Exemplary reporters comprise at least onedye, such as a fluorescent dye or an energy transfer pair, and/or atleast one oligonucleotide. Exemplary reporters for nucleic acidamplification assays may include a probe and/or an intercalating dye(e.g., SYBR Green, ethidium bromide, etc.).

In some embodiments, one or more PCR reagents can be combined to form acomposition or a kit. In some embodiments, a composition can include anysuitable PCR reagents that are required for carrying out anamplification reaction including the novel thermally-labile smallmolecule DNA polymerase inhibitors of the present disclosure. Forexample, compositions can include a DNA polymerase inhibitor of thepresent disclosure and one or more PCR reagents such as one or more of aprimer or pair of primers for amplification of a nucleic acid target, aprobe and/or dye to enable detection of amplification, a ligase, apolymerase, nucleotides (dNTPs and/or NTPs), divalent magnesium ions, orany combination thereof, among other reagents that would be recognizedby one of ordinary skill in the art based on the present disclosure.These various combinations of PCR reagents, including the novelthermally-labile small molecule DNA polymerase inhibitors, can also beincluded in a kit used to carry out PCR reactions including hot-startPCR reactions.

In accordance with the embodiments provided herein, concentrations ofthe PCR reagents described above can vary, depending on specificreaction conditions and reagents used, as well as the desired DNA targetto be amplified. One of skill in the art would readily recognize thatany specific concentrations or concentration ranges provided herein forany PCR reagents, including concentration ranges pertaining to the DNApolymerase inhibitors of the present disclosure, will vary depending onthe specific reaction conditions and reagents used and are not meant tobe limiting.

4. MATERIALS AND METHODS

Embodiments of the present disclosure include various compositions andmethods used to carry out PCR reactions, including, but not limited to,hot-start PCR reactions. Generally, PCR reactions involve a processreplication or forming a copy (e.g., a direct copy and/or acomplementary copy) of a nucleic acid or a segment thereof. Replicationreactions generally involves an enzyme, such as a polymerase and/or aligase, among others. The nucleic acid and/or segment replicated is atemplate (and/or a target) for replication. PCR reactions also generallyinvolve a process of amplification, or a reaction in which replicationoccurs repeatedly over time to form multiple copies of at least onesegment of a template molecule. Amplification may generate anexponential or linear increase in the number of copies as amplificationproceeds. Typical amplifications produce a greater than 1,000-foldincrease in copy number and/or signal. Exemplary amplification reactionsfor the assays disclosed herein may include the polymerase chainreaction (PCR) or ligase chain reaction (LCR), each of which is drivenby thermal cycling. Thermal cycling generally involves cycles of heatingand cooling a reaction mixture to perform successive rounds ofdenaturation (melting), annealing, and extension. Additionally oralternatively, assays provided herein may use other amplificationreactions, which may be performed isothermally, such as branched-probeDNA assays, cascade-RCA, helicase-dependent amplification, loop-mediatedisothermal amplification (LAMP), nucleic acid based amplification(NASBA), nicking enzyme amplification reaction (NEAR), PAN-AC, Q-betareplicase amplification, rolling circle replication (RCA),self-sustaining sequence replication, strand-displacement amplification,and the like. Amplification may utilize a linear or circular template.

Amplification may be performed with any suitable reagents, as describedabove, including the novel thermally-labile small molecule DNApolymerase inhibitors of the present disclosure. Amplification may beperformed, or tested for its occurrence, in an amplification mixture,which is any composition capable of generating multiple copies of anucleic acid target molecule, if present, in the composition. Anamplification mixture may include any combination of at least one primeror primer pair, at least one probe, at least one replication enzyme(e.g., at least one polymerase, such as at least one DNA and/or RNApolymerase, and/or at least one ligase), and/or deoxynucleotide (and/ornucleotide) triphosphates (dNTPs and/or NTPs), among others.

As described further herein, hot-start PCR amplification generallyincludes amplification reactions (e.g., polymerization and/or ligation)that do not amplify a specific target without also, for example,amplifying non-specific targets, until an elevated temperature isreached. The elevated temperature may be at least about the annealingtemperature for the primers to reduce non-specific amplification. Insome cases, hot-start PCR allows for the amplification of a giventarget, or the amplification of an increased yield of a given target andis not necessarily required to reduce amplification of non-specifictargets. In other cases, the nuclease activity of a polymerase enzymecan cause degradation of primers and templates (e.g., resulting in theremoval of fluorescent dyes, or decrease in primer or probe length);and, in some cases, hot-start PCR can reduce or prevent undesirednuclease activity.

Generally, PCR includes any nucleic acid amplification reaction thatrelies on alternating cycles of heating and cooling (i.e., thermalcycling) to achieve successive rounds of replication. PCR may beperformed by thermal cycling between two or more temperature set points,such as a higher melting (denaturation) temperature and a lowerannealing/extension temperature, or among three or more temperature setpoints, such as a higher melting temperature, a lower annealingtemperature, and an intermediate extension temperature, among others.PCR may be performed with a heat-stable polymerase, such as Taq DNApolymerase (e.g., wild-type enzyme, a Stoffel fragment, FastStartpolymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase,Vent polymerase, or a combination thereof, among others described above.PCR generally produces an exponential increase in the amount of aproduct amplicon over successive cycles.

Any suitable PCR methodology or combination of methodologies may beutilized in the embodiments disclosed herein, such as allele-specificPCR, assembly PCR, asymmetric PCR, digital PCR, endpoint PCR, hot-startPCR, in situ PCR, intersequence-specific PCR, inverse PCR, linear afterexponential PCR, ligation-mediated PCR, methylation-specific PCR,miniprimer PCR, multiplex ligation-dependent probe amplification,multiplex PCR, nested PCR, overlap-extension PCR, polymerase cyclingassembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR,single-cell PCR, solid-phase PCR, thermal asymmetric interlaced PCR,touchdown PCR, universal fast walking PCR, or any combination thereof,among others.

In accordance with embodiments described above, various materials andmethods used to investigate the thermal labile small molecule polymeraseinhibitors of the present disclosure are described below.

Taq and Thermal Degradable Detergent Titrations. FIG. 9 includes arepresentative table of the results of various hot-start PCR reactions(Corynephage omega gene target) performed with polymerase inhibitorcompound #7124 and polymerase enzyme at the various concentrationsshown. Ranges included from about 0.375 to 1.25 U Taq/25 μl reaction andfrom 100-250 μM of compound #7124. FIG. 10 includes a representativetable of the results of various hot-start PCR reactions performed withpolymerase inhibitor compound #7261 and polymerase enzyme at the variousconcentrations shown. Ranges included from about 0.375 to 1.25 U Taq/25μl reaction, and from 50-175 μM of compound #7216. (See Table 1 below.)

TABLE 1 Taq Level in Figure U Taq/25 ul reaction U Taq/ul reaction 0.375U 0.375 U/25 ul reaction  0.0056 U/ul 0.5 U  0.5 U/25 ul reaction 0.01U/ul 0.625 U 0.625 U/25 ul reaction  0.025 U/ul 1.25 U 1.25 U/25 ulreaction 0.05 U/ul 2.5 U  2.5 U/25 ul reaction 0.1 U/ul 3.75 U 3.75 U/25ul reaction 0.15 U/ul 5 U   5 U/25 ul reaction 0.2 U/ul 6.25 U 6.25 U/25ul reaction 0.25 U/ul

Testing of Human Genomic Templates. FIG. 11 includes a representativeimage of an agarose gel used to evaluate the CCR5 gene amplificationproducts of various hot-start PCR reactions performed with and withouttwo different polymerase inhibitors (compound #7124 and compound #7261)and using human genomic DNA as a template. The target amplicon is 500bp, and without the use of hot-start, there is a significant amount ofprimer dimer. With compound #7124 and compound #7261, the 500 bp yieldincreased, but primer dimer amounts remained (compare to GoTaq® DNAPolymerase treatment for no hot-start and GoTaq® Hot-Start DNAPolymerase with an antibody-mediated hot-start). Reaction components areprovided in Table 2 below; the following cycling methods were used:[(22° C. for 90 min, 94° C. for 2 min) 1 cycle, (92° C. for 30 sec, 68°C. for 2 min) 45 cycles, (72° C. for 5 min) 1 cycle, 4° C. soak].

FIG. 12 includes a representative image of an agarose gel used toevaluate the Human Dystrophin Alu-72 gene amplification products ofvarious hot-start PCR reactions performed with and without two differentpolymerase inhibitors (compound #7124 and compound #7261) and usinghuman genomic DNA as a template. The target amplicon is 320 bp, andwithout hot-start, there is less yield. With compound #7124 and compound#7261, the 320 bp yield increased, and at certain concentrations,secondary products (seen as a smear) dramatically decreased. Reactioncomponents are provided in Table 2 below; the following cycling methodswere used: [(22° C. for 90 min, 94° C. for 2 min) 1 cycle, (94° C. for50 sec, 59° C. for 50 sec, 68° C. for 2 min) 42 cycles, 4° C. soak].

TABLE 2 Component Corynephage omega gene CCR5 Hot-Start HD Alu 7-2Hot-Start Small Molecule Inhibitor Taq 0.025 U/ul reaction 0.025 U/ulreaction 0.025U/ul reaction Primers 0.4 uM  0.4 uM  0.2 uM  Detergent:PPD (Promega 0.0025%, variable 0.0025% 0.0025% Proprietary Detergent)when did Taq titration expts FIG. 9 & 10 Detergent: PPD (Promega0.0025%, variable 0.0025% 0.0025% Proprietary Detergent) when did Taqtitration expts FIG. 9 & 10 dNTPs (each) 0.2 mM 0.2 mM 0.2 mM Mg   2 mM  2 mM   2 mM Template: plasmid 0.5 ng/25 ul Rxn NA NA Template: HumanNA 100 pg/ul (2.5 ng), 10 pg/ul (500 pg), 1 pg/ul Genomic 10 pg/ul (250pg), 1 pg/ul (50 pg), 0.1 pg/ul (5 pg) (25 pg)

5. EXAMPLES

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the presentdisclosure described herein are readily applicable and appreciable andmay be made using suitable equivalents without departing from the scopeof the present disclosure or the aspects and embodiments disclosedherein. Having now described the present disclosure in detail, the samewill be more clearly understood by reference to the following examples,which are merely intended only to illustrate some aspects andembodiments of the disclosure, and should not be viewed as limiting tothe scope of the disclosure. The disclosures of all journal references,U.S. patents, and publications referred to herein are herebyincorporated by reference in their entireties.

The present disclosure has multiple aspects, illustrated by thefollowing non-limiting examples.

Example 1 Compound Syntheses

Polymerase inhibitors can be made by the following general procedures.

Aldehyde (1 equivalent) reacted with the desired Grignard reagent (1.2equivalent) in dry THF at 0° C. The mixture was stirred at RT overnight.The reaction was quenched by adding 20 mL of water and then 100 mL ofether added. The resulting mixture was acidified by 2N HCl until theprecipitates disappeared. The aqueous layer was extracted three timeswith ether, and the combined organic layer was washed with water anddried over Na₂SO₄. The resulting alcohol was purified by silica columnusing heptane/ethyl acetate as the eluent.

To the solution of the above alcohol (1 equivalent) andp-nitrophenylchloroformate (2 equivalent) in 20 mL dry THF, pyridine (3equivalent) was added slowly at 0° C. The reaction mixture was stirredfor 1 hour. After removing the solvent, the residue was dissolved inheptane. The insoluble precipitate was filtered out. After removing thesolvent of filtrate, the crude p-nitrophenylcarbonate was used directly.

To the solution of p-nitrophenylcarbonate (1 equivalent), the desiredamine TBA salt (3 equivalent) was added. The mixture was stirred for 1hour. The solid was removed by filtration. After removal of the solvent,the compound was dissolved in DCM and purified by silica column usingheptane/ethyl acetate to DCM/MeOH as solvent. The viscous solid wasobtained. Bisodium resin was washed with deionized water 3-4 times. Thecompound was loaded to resin column for ion-exchange. The compound wasrinsed out quickly, and the desired fractions were collected andlyophilized.

Compound structures are listed below.

Sodium3-((((1-(4-methoxyphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7124). ¹H NMR (d⁶-DMSO, δ ppm): 7.25 (d, 2H), 7.15 (t, 1H), 6.82 (d,2H), 5.48 (t, 1H, CH), 3.73 (s, 3H, OCH3), 2.93 (m, 2H, CH2), 2.38 (t,2H, CH2), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.75 (t, CH3,3H). MS (m/e) [M⁻] (C₂₄H₄₀NO₆S⁻): calculated 470.26, observed 470.3.

Sodium3-((((1-(5-chlorofuran-2-yl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7261). ¹H NMR (d⁶-DMSO, δ ppm): 7.50 (t, 1H, NH), 6.52 (d, 1H), 6.41(d, 1H), 5.55 (t, 1H, CH), 3.01 (q, 2H, CH2), 2.32 (t, 2H, CH2), 1.81(q, 2H, CH2), 1.62 (m, 2H, CH2), 1.1-1.4 (m, CH2, 20H), 0.8 (t, CH3,3H). MS (m/e) [M⁻] (C₂₁H₃₅ClNO₆S): calculated 464.19, observed 464.3.

Sodium3-((((1-(2,3-dihydrobenzofuran-5-yl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7489). ¹H NMR (d⁶-DMSO, δ ppm): 7.20 (s, 1H), 7.12 (t, 1H), 6.95 (d,1H), 6.65 (d, 1H), 5.48 (t, 1H, CH), 4.5 (t, 2H, CH2), 3.20 (t, 2H,CH2), 2.36 (m, 2H, CH2), 2.38 (t, 2H, CH2), 1.5-1.8 (m, 4H, CH2),1.1-1.4 (m, CH2, 20H), 0.80 (t, CH3, 3H). MS (m/e) [M⁻](C₂₄H₄₀NO₆S⁻):calculated 482.26, observed 482.5.

Sodium3-((((1-(chroman-6-yl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7490). ¹H NMR (d⁶-DMSO, δ ppm): 7.15 (t, 1H), 6.98 (m, overlap, 2H),6.65 (d, 1H), 5.48 (t, 1H, CH), 4.17 (t, 2H, CH2), 2.91 (m, 2H, CH2),2.75 (t, 2H, CH2), 2.37 (t, 2H, CH2), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m,CH2, 20H), 0.80 (t, CH3, 3H). MS (m/e) [M⁻] (C₂₆H₄₂NO₆S⁻): calculated496.27, observed 496.6.

Sodium3-((((1-(4-methoxy-2-methylphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7487). ¹H NMR (d⁶-DMSO, δ ppm): 7.21 (d, 1H), 7.10 (t, 1H), 6.73 (d,1H), 6.62 (s, br, 1H), 5.61 (t, 1H, CH), 3.68 (s, 3H, OCH3), 2.94 (m,2H, CH2), 2.37 (t, 2H, CH2), 2.30 (s, 3H, CH3), (t, 2H, CH2), 1.5-1.8(m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3, 3H). MS (m/e)[M⁻](C₂₅H₄₂NO₆S⁻): calculated 484.27, observed 484.5.

Sodium3-((((1-(4-methoxy-3-methylphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7488). ¹H NMR (d⁶-DMSO, δ ppm): 7.0-7.2 (m, 3H), 6.83 (d, 1H), 5.63 (t,1H, CH), 3.63 (s, 3H, OCH3), 2.94 (m, 2H, CH2), 2.37 (t, 2H, CH2), 2.12(s, 3H, CH3), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3,3H). MS (m/e) [M⁻](C₂₅H₄₂NO₆S⁻): calculated 484.27, observed 484.5.

Sodium3-((((1-(4-methoxy-2,3-dimethylphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate (7491). ¹H NMR (d⁶-DMSO, δ ppm): 7.31 (t, 1H, NH),7.11 (d, 1H), 6.73 (d, 1H), 5.67 (t, 1H, CH), 3.77 (s, 3H, OCH3), 2.91(m, 2H, CH2), 2.37 (t, 2H, CH2), 2.19 (s, 3H, CH3), 2.12 (s, 3H, CH3),1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3, 3H). MS (m/e)[M⁻] (C₂₆H₄₄NO₆S⁻): calculated 498.29, observed 498.5.

Sodium3-((((1-(4-methoxy-2,5-dimethylphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate (7493). ¹H NMR (d⁶-DMSO, δ ppm): 7.13 (t, 1H, NH),7.0 (s, 1H), 6.67 (s, 1H), 5.67 (t, 1H, CH), 3.73 (s, 3H, OCH3), 2.95(m, 2H, CH2), 2.35 (t, 2H, CH2), 2.29 (s, 3H, CH3), 2.14 (s, 3H, CH3),1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.79 (t, CH3, 3H). MS (m/e)[M⁻] (C₂₆H₄₄NO₆S⁻): calculated 498.29, observed 498.5.

Sodium3-((((1-(4-methoxy-2,6-dimethylphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate (7494). ¹H NMR (d⁶-DMSO, δ ppm): 7.13 (t, 1H, NH),6.51 (s, 2H), 5.67 (t, 1H, CH), 3.68 (s, 3H, OCH3), 2.95 (m, 2H, CH2),2.38 (m, overlap, 8H, 2CH3+CH2), 1.5-2.0 (m, 4H, CH2), 1.1-1.4 (m, CH2,20H), 0.81 (t, CH3, 3H). MS (m/e) [M⁻] (C₂₆H₄₄NO₆S⁻): calculated 498.29,observed 498.5.

Sodium3-((((1-(4-methoxy-2,3,6-trimethylphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate (7640). ¹H NMR (d⁶-DMSO, δ ppm): 7.13 (t, 1H, NH),6.61 (s, 1H), 5.72 (t, 1H, CH), 3.72 (s, 3H, OCH3), 2.94 (m, 2H, CH2),2.38 (m, overlap, 5H, CH3+CH2), 2.27 (s, 3H, CH3), 2.09 (s, 3H, CH3),1.5-2.0 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3, 3H). MS (m/e)[M⁻] (C₂₇H₄₆NO₆S⁻): calculated 512.31, observed 512.5.

Sodium3-((((1-(5-bromofuran-2-yl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7484). ¹H NMR (d⁶-DMSO, δ ppm): 7.25 (t, 1H, NH), 6.51 (d, 1H), 6.48(d, 1H), 5.53 (t, 1H, CH), 2.97 (m, 2H, CH2), 2.32 (t, 2H, CH2), 1.82(q, 2H, CH2), 1.63 (m, 2H, CH2), 1.1-1.4 (m, CH2, 20H), 0.8 (t, CH3,3H). MS (m/e) [M⁻] (C₂₅H₃₅BrNO₆S): calculated 508.14, observed 508.8.

Sodium3-((((1-(3,4-dimethoxyphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7486). ¹H NMR (d⁶-DMSO, δ ppm): 7.21 (t, 1H, NH), 6.7-7.0 (m, 3H), 5.48(t, 1H, CH), 3.73 (m, 6H, OCH3), 2.93 (m, 2H, CH2), 2.38 (t, 2H, CH2),1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.75 (t, CH3, 3H). MS (m/e)[M⁻] (C₂₅H₄₂NO₇S⁻): calculated 500.67, observed 500.3.

Sodium3-((((1-(4-methoxyphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7124). ¹H NMR (d⁶-DMSO, δ ppm): 7.25 (d, 2H), 7.15 (t, 1H), 6.82 (d,2H), 5.48 (t, 1H, CH), 3.73 (s, 3H, OCH3), 2.93 (m, 2H, CH2), 2.38 (t,2H, CH2), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.75 (t, CH3,3H). MS (m/e) [M⁻] (C₂₄H₄₀NO₆S⁻): calculated 470.26, observed 470.3.

Sodium3-((((1-(4-isopropoxyphenyl)tridecyl)oxy)carbonyl)amino)propane-1-sulfonate(7495). ¹H NMR (d⁶-DMSO, δ ppm): 7.25 (d, 2H), 7.18 (t, 1H, NH), 6.81(d, 2H), 5.48 (t, 1H, CH), 4.61 (m, 1H, CH), 2.93 (m, 2H, CH2), 2.39 (t,2H, CH2), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.75 (t, CH3,3H). MS (m/e) [M⁻] (C₂₆H₄₄NO₆S⁻): calculated 498.70, observed 498.5.

4-((((1-(4-methoxyphenyl)tridecyl)oxy)carbonyl)amino)butanoic acid(7365). ¹H NMR (d⁶-DMSO, δ ppm): 7.25 (d, 2H), 6.82 (d, 2H), 5.67 (t,1H, CH), 4.85 (s, br, 1H), 3.69 (s, 3H, OCH3), 3.2 (m, 2H, CH2), 2.38(t, 2H, CH2), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.78 (t, CH3,3H). MS (m/e) [M-H] (C₂₅H₄₁NO₅): calculated 435.3, observed 434.5.

(((1-(4-methoxyphenyl)tridecyl)oxy)carbonyl)glutamic acid (7366). ¹H NMR(d⁶-DMSO, δ ppm): 7.21 (d, 2H), 6.87 (d, 2H), 5.5-5.7 (m, 1H), 4.1-4.3(m, 2H), 3.69 (s, 3H, OCH3), 1.5-1.8 (m, 4H, CH2), 1.1-1.4 (m, CH2,20H), 0.83 (t, CH3, 3H). MS (m/e) [M⁻] (C₂₆H₄₁NO₇): calculated 479.3,observed 478.5.

(((1-(4-methoxyphenyl)tridecyl)oxy)carbonyl)aspartic acid (7367). ¹H NMR(d⁶-DMSO, δ ppm): 7.25 (d, 2H), 6.87 (d, 2H), 5.5-5.7 (m, 1H), 4.62 (m,1H), 3.81 (s, 3H, OCH3), 2.7-3.2 (m, 2H), 1.5-2.0 (m, 4H, CH2), 1.1-1.4(m, CH2, 20H), 0.83 (t, CH3, 3H). MS (m/e) [M-H] (C₂₅H₃₉NO₇): calculated465.27, observed 464.3.

4-((((1-(5-chlorofuran-2-yl)tridecyl)oxy)carbonyl)amino)butanoic acid(7437). ¹H NMR (d⁶-DMSO, δ ppm): 7.26 (t, 1H, NH), 6.52 (d, 1H), 6.43(d, 1H), 5.55 (t, 1H, CH), 2.95 (m, 2H, CH2), 2.37 (t, 2H, CH2), 1.81(q, 2H, CH2), 1.63 (m, 2H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3,3H). MS (m/e) [M⁻] (C₂₂H₃₆ClNO₅): calculated 429.23, observed 428.4.

4-((((1-(5-chlorofuran-2-yl)tridecyl)oxy)carbonyl)amino)glutamic acid(7439). ¹H NMR (d⁶-DMSO, δ ppm): 7.53 (t, 1H, NH), 6.53 (d, 1H), 6.44(d, 1H), 5.55 (t, 1H, CH), 3.91 (m, 2H), 2.28 (m, 2H, CH2), 1.6-2.2 (m,4H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3, 3H). MS (m/e) [M⁻](C₂₃H₃₆ClNO₇): calculated 473.22, observed 472.4.

4-((((1-(5-chlorofuran-2-yl)tridecyl)oxy)carbonyl)amino)aspartic acid(7438). ¹H NMR (d⁶-DMSO, δ ppm): 7.59 (t, 1H, NH), 6.53 (m, 1H), 6.44(m, 1H), 5.55 (t, 1H, CH), 4.21 (m, 2H), 2.5-2.8 (m, 2H, CH2), 1.72 (m,2H, CH2), 1.1-1.4 (m, CH2, 20H), 0.81 (t, CH3, 3H). MS (m/e) [M⁻](C₂₃H₃₆ClNO₇): calculated 459.22, observed 458.4.

Example 2 DNA Polymerase Inhibitor #7124 and Derivatives

To demonstrate that the thermally-labile small molecule polymeraseinhibitors of the present disclosure can provide hot-startamplification, a 1.5 kb fragment of the Corynephage omega gene fromplasmid DNA was amplified. When carried out using hot-start conditions,the polymerase (e.g., Taq polymerase) was inhibited at lowertemperatures, but activated at the start of amplification whentemperatures increased to denature the DNA (e.g., 95° C.); theamplification produced a single product approximately 1.5 kb in size.When carried out using non-hot-start conditions, the DNA polymerase wasnot inhibited at lower temperatures, and amplification produced aproduct approximately 400 bp in size (among other secondary products),and the 1.5 kb fragment was transiently present. To test the ability ofthe thermally-labile small molecule polymerase to inhibit DNA polymerase(e.g., Taq DNA polymerase) activity, the amplification reactions wereincubated at 22° C. for 1.5 to 6 hours or longer prior to performing PCRamplification.

The amplifications were set up at room temperature. The thermally-labilesmall molecule polymerase inhibitor(s) were titrated (concentrationswill depend on exact compound, enzyme being inhibited, detergentconcentration, and other buffer conditions) into reactions. For theseexperiments, the following composition were used: 1× GoTaq® ColorlessFlexi Buffer, 2.5 mM MgCl₂, 200 μM each dNTP, 0.4 μM each primer, 0.025U/μl GoTaq® DNA Polymerase, 500 pg plasmid DNA and nuclease-free waterto bring it to a 25 μl reaction. “No small molecule inhibitor”, “notemplate control” (NTC), and “positive hot-start” (using GoTaq® DNApolymerase with an antibody-mediated hot-start) control reactions wereassembled. The reactions were inserted into a room temperature thermalcycler, and the following cycling protocol was used: 1 cycle (22° C. for1.5 to 6 hours, 95° C. for 2 minutes), 30 cycles (93° C. for 15 seconds,54° C. for 30 seconds, 72° C. for 1 minute), 1 cycle (72° C. for 5minutes), and 4° C. soak. Once cycling was complete, PCR products wereseparated and visualized on a 1% agarose gel with ethidium bromidestaining and UV-light illumination. A camera was used to record theimage of the gel.

As expected, hot-start control reactions (using antibody-mediatedhot-start DNA polymerase) and reactions with small molecule inhibitor(s)exhibiting hot-start ability amplified a single 1.5 kb product. In the“no small molecule inhibitor control” reactions and reactions with smallmolecule inhibitor(s) not exhibiting hot-start ability, a 400 bp productwas amplified (Other secondary products and a 1.5 kb product may or maynot be present)). In the “no template” control, no amplificationoccurred.

FIGS. 1A-1B include representative images of agarose gels used toevaluate the amplification products of various hot-start PCR reactionsperformed with and without one of two different polymerase inhibitors ofthe present disclosure (compound #7124 in FIG. 1A; compound #7261 inFIG. 1B). FIG. 1A includes hot-start PCR amplification results usingcompound #7124 at the various concentrations listed. As demonstrated,the use of compound #7124 prevented non-specific DNA amplification (˜400kb product) while facilitating amplification of the desired DNA target(˜1.5 kb). Although exact concentrations will vary depending on specificreaction conditions and reagents, use of compound #7124 at aconcentration ranging from about 100 μM to about 150 μM facilitated waseffective, and specific amplification of the desired DNA target wasachieved.

FIG. 3 includes a representative table of the results of varioushot-start PCR reactions (amplifying the 1.5 kb fragment of theCorynephage omega gene target from plasmid DNA) performed with differentpolymerase inhibitors that are derivatives of compound #7124 (i.e.compound #7489 and compound #7490). As demonstrated, the use of compound#7489 prevented non-specific DNA amplification and facilitated specificamplification of the desired DNA target at concentrations ranging fromabout 75 μM to about 175 μM. The use of compound #7490 preventednon-specific DNA amplification and facilitated specific amplification ofthe desired DNA target at concentrations ranging from about 100 μM toabout 125 μM.

FIG. 4 includes a representative table of the results of varioushot-start PCR reactions (amplifying the 1.5 kb fragment of theCorynephage omega gene target from plasmid DNA) performed with differentpolymerase inhibitors that are derivatives of compound #7124 (i.e.compound #7487 and compound #7488). As demonstrated, the use of compound#7487 prevented non-specific DNA amplification and facilitated specificamplification of the desired DNA target at concentrations ranging fromabout 75 μM to about 150 μM. The use of compound #7488 preventednon-specific DNA amplification and facilitated specific amplification ofthe desired DNA target at concentrations ranging from about 75 μM toabout 100 μM.

FIG. 5 includes a representative table of the results of varioushot-start PCR reactions (amplifying the 1.5 kb fragment of theCorynephage omega gene target from plasmid DNA) performed with differentpolymerase inhibitors that are derivatives of compound #7124 (i.e.compound #7491 and compound #7493). As demonstrated, the use of compound#7491 prevented non-specific DNA amplification and facilitated specificamplification of the desired DNA target at concentrations ranging fromabout 75 μM to about 125 μM. The use of compound #7493 preventednon-specific DNA amplification and facilitated specific amplification ofthe desired DNA target at concentrations ranging from about 100 μM toabout 125 μM.

FIG. 6 includes a representative table of the results of varioushot-start PCR reactions (amplifying the 1.5 kb fragment of theCorynephage omega gene target from plasmid DNA) performed with differentpolymerase inhibitors that are derivatives of compound #7124 (i.e.compound #7486 and compound #7495). As demonstrated, the use of compound#7486 prevented non-specific DNA amplification and facilitated specificamplification of the desired DNA target at concentrations ranging fromabout 125 μM to about 175 μM. The use of compound #7495 preventednon-specific DNA amplification and facilitated specific amplification ofthe desired DNA target at concentrations ranging from about 50 μM toabout 75 μM.

With respect to FIGS. 2-6 , “No HS” designates reactions that did notprevent amplification of non-specific products; “HS” designatesreactions that prevented amplification of non-specific products andfacilitated amplification of specific products; and “No Amp” designatesno amplification of specific or non-specific products. Amplificationyield assessment is provided in parentheses as: “++” (good yield similarto hot-start control); “+” (moderate yield), and “low” (low yield).

Example 3 DNA Polymerase Inhibitor #7261 and Derivatives

The same methods described in Example 2 above were used to test theactivity of DNA polymerase inhibitor #7261 and its derivatives. FIG. 1Bincludes hot-start PCR amplification results (amplifying the 1.5 kbfragment of the Corynephage omega gene target from plasmid DNA) usingcompound #7261 at the various concentrations listed. As demonstrated,the use of compound #7261 prevented non-specific DNA amplification (˜400kb product) while facilitating amplification of the desired DNA target(˜1.5 kb). Although exact concentrations will vary depending on specificreaction conditions and reagents, use of compound #7261 at aconcentration ranging from about 75 μM to about 100 μM facilitatedeffective and specific amplification of the desired DNA target.

FIG. 2 includes a representative table of the results of varioushot-start PCR reactions (amplifying the 1.5 kb fragment of theCorynephage omega gene target from plasmid DNA) performed with differentpolymerase inhibitors that are derivatives of compound #7261 (i.e.,compound #7437, compound #7438, and compound #7439). As demonstrated,the use of compound #7437 prevented non-specific DNA amplification andfacilitated specific amplification of the desired DNA target atconcentrations ranging from about 100 μM to about 150 μM. The use ofcompound #7438 prevented non-specific DNA amplification and facilitatedspecific amplification of the desired DNA target at concentrationsranging from about 25 μM to about 200 μM. The use of compound #7439prevented non-specific DNA amplification and facilitated specificamplification of the desired DNA target at concentrations ranging fromabout 50 μM to about 225 μM. In particular, potency of DNA polymerase#7261 inhibitors was enhanced from about 75 μM to from about 25 μM toabout 150 μM or greater for #7438 and #7439.

Example 4 Evaluation of Polymerase Inactivation at DifferentTemperatures

Activity assays were used to test the concentrations of small moleculepolymerase inhibitors in specific reaction conditions to determineinhibition efficacy. Corresponding IC₅₀ values were also determined forthe compounds. Activity assays were also used to test the temperaturesor temperature ranges at which the reactions are inhibited by thecompounds.

Exemplary activity assays can be carried out according to a variety ofmethods. For example, a DNA polymerase activity assay monitoringradioactive incorporation where “activated” calf thymus or salmon spermDNA is used as the DNA substrate. Along with the DNA substrate, thereactions minimally contain a buffer (e.g., GoTaq® buffer), magnesium,dNTPs, and polymerase. Reactions were stopped, and DNA was precipitatedby ice-cold TCA (tricholoracetic acid), incubated on ice for at least 10minutes, filtered using GF/C filters, and radioactive incorporation inprecipitable DNA (on filter) measured by scintillation counting. (See,e.g., Apospian & Kornberg. (1962) JBC 237: 519-525; Chien et al. (1976)J. Bact. 127: 1550-1557.)

A primer extension DNA polymerase activity assay monitoring radioactiveincorporation, wherein single-stranded DNA (e.g., M13) and primersubstrate was used as the DNA substrate. The primer and template wereannealed, and reactions minimally contain buffer (e.g., GoTaq® buffer),magnesium, dNTPs, and polymerase. Reactions were stopped, and DNAprecipitated by ice-cold TCA, incubated on ice for at least 10 minutes,filtered using GF/C filters, and radioactive incorporation inprecipitable DNA on filter measured by scintillation counting. (See,e.g., Longley & Mosbaugh. (1991) Biochemistry 30: 2655-2664.)

FIG. 7 includes a representation of a polymerase activity assay carriedout at 22° C., where an inhibitor (i.e. compound #7124) is titrated.FIG. 7 is an example of an activity assay where IC₅₀ can be determined.Compound 7124 did not inhibit Taq DNA Polymerase at low concentrations;however, as concentration increased, inhibition begins and ultimatelyreaches 100% inhibition. From this graph, an IC₅₀ was calculated to beabout 150 μM. Activated calf thymus DNA activity assay was used in theseexperiments. Assays similar to this can be used to determine otherconditions under which enzyme inhibition occurs (e.g., temperatures,buffer conditions, and different DNA polymerases).

FIG. 8 includes a representative table of results of IC₅₀ values fordifferent polymerase inhibitors when incubated at 22° C. (i.e. compound#7124, compound #7126, compound #7127, compound #7123, compound #7125,compound #6966, and SDS). FIG. 8 is an example of IC₅₀ information for avariety of compounds. To determine the IC₅₀ values, experiments similarto those done in FIG. 7 were performed. Activated calf thymus DNA methodwas used for these experiments.

Example 5 Evaluation of Activation of Enzymes by Temperature

To test activation temperatures and times to determine when activity isrestored, activity assay reactions can be assembled with and withoutsmall molecule polymerase inhibitors (as described further above).Reactions start at low temperature (e.g., 22° C. or 37° C.) for a periodof time. Temperatures of the reactions can be increased to a hightemperature (e.g., >90° C.) and incubated for a given amount of time toallow for enzyme activation. The reaction temperatures can be decreasedto 68-79° C. and incubated for 15 minutes. Reactions can be stopped,samples processed, and activity calculated to determine the amount ofenzyme activity that can be recovered.

In accordance with these methods, PCR can be used to test activation ofpolymerase enzymes and to determine the most effective conditions foreach small molecule inhibitor, for example, by adjusting the temperatureand time of the initial denaturation step (e.g., Corynebacterium omegagene, or a target from human genomic DNA).

Example 6 Real-Time Inhibition and Activation Conditions

In accordance with the methods of the present disclosure, experimentscan also be conducted using a real-time extension rate activity assaymethod to measure nucleotide incorporation of a DNA polymerase. Forexample, a primer extension assay can include a process by whichextension is monitored on a real-time PCR instrument using non-covalentDNA dyes such as BRYT™ Green or SYBR® Green and an oligonucleotide DNAsubstrate. (See, e.g., Montgomery & Wittwer (2014), Clinical Chemistry60(2):334-340). Activity assay reactions can be assembled with andwithout a small molecule polymerase inhibitor. The reactions minimallyinclude buffer (e.g., GoTaq® buffer), magnesium, dNTPs, DNA substrate,and polymerase.

In one embodiment of the assay, the reactions can be monitored todetermine the temperature at which the enzyme starts to be activated.Reactions can be incubated at a low temperature (e.g., 22° C., 37° C.,55° C., etc.) to a high temperature (68° C., 72° C., 79° C., etc.) for agiven period of time while measuring extension rate to assess inhibitionof the polymerase.

In another embodiment of the assay, the temperature and times needed foractivation can be investigated. Reactions can be incubated at a lowtemperature (e.g., 22° C. or 37° C.) for a period of time whilemonitoring the activity. Temperatures of the reactions can be increasedto a high temperature (e.g., >90° C.) and incubated for a given amountof time to try to activate the enzyme. The reaction temperatures canthen be decreased to 68-79° C. and incubated as in the standard activityassay to measure the enzyme activity recovered.

Example 7 Evaluation of Compounds for Inhibition of Nuclease ActivityAssociated with DNA Polymerases

Many DNA polymerases have associated domains with nuclease activity(e.g., Taq has a 5′ nuclease and Pfu has a 3′ to 5′ proof-readingnuclease). Both the 5′ nuclease and the 3′ to 5′ nuclease activities canbe problematic for PCR. For example, the 5′ nuclease activity can removedyes on the 5′ end of primers if the appropriate structure is formedwith the DNA in the reaction during set up. Also, for example, the 3′ to5′ nuclease activity can degrade primers and template in the reactionduring setup. These troublesome side reactions can also be inhibited bythe small molecule polymerase inhibitors of the present disclosure.

To study the ability of the small molecule polymerase inhibitors of thepresent disclosure to inhibit nuclease activities, a 5′ nucleaseactivity assay (for an enzyme such as Taq) can be performed using a 5′fluorescently dye-labeled bifurcated duplex DNA substrate. (See, e.g.,Lyamichev et al. (1993) Science 260: 778-783; Lyamichev (1999) PNAS96:6143-6148; Ceska & Sayers (1998) TIBS: 331-336). The DNA substratecan be annealed and combined with reaction components including buffer(e.g., GoTaq® buffer), magnesium, and a nuclease or a polymerase with anuclease domain. Reaction can be stopped with EDTA and run on acapillary electrophoresis instrument to determine amount of cut anduncut DNA substrate.

Additionally, a 3′ to 5′ exonuclease activity assay (for a proofreadingpolymerase such as Pfu) can be performed using a 3′-radiolabeledduplexed DNA as a substrate. The DNA substrate can be combined withreaction components including buffer (e.g., GoTaq® buffer), magnesium,and a nuclease or a polymerase with a nuclease domain. Reactions can bestopped by EDTA, and DNA can be precipitated by ice-cold TCA andincubated on ice for at least 10 minutes. Precipitable DNA can bepelleted by centrifugation and released non-precipitable DNA fromradioactively labeled 3′ end can be measured by scintillation counting.(See, e.g., Chase & Richardson. (1974) JBC 249: 4545-4552; Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition), pp.10.51-52).

Example 8 Variations of Methods

As would be appreciated by one of ordinary skill in the art based on thepresent disclosure, the small molecule polymerase inhibitors of thepresent disclosure can be used to inhibit the activity of other enzymes,or activities of associated domains, such as, but not limited to,ligase, reverse transcriptase, and RNase H. Activity of these enzymescan be investigated in the context of PCR, rtPCR, or other DNA/RNAamplification methods.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the disclosure, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the disclosure, may be made withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A compound of formula (I):

or a salt thereof, wherein: A is selected from aryl, heteroaryl, andheterocyclyl, each of which may be optionally substituted with 1, 2, or3 substituents; R¹ is C₆-C₂₀ alkyl; R² is selected from hydrogen and—COOH; and n is 1 or
 2. 2. The compound of claim 1, or a salt thereof,wherein A is phenyl that is unsubstituted or substituted with 1, 2, or 3substituents independently selected from C₁-C₄ alkyl, C₁-C₄ alkoxy, andhalo.
 3. The compound of claim 1, or a salt thereof, wherein A is a 5-or 6-membered monocyclic heteroaryl that is unsubstituted or substitutedwith 1, 2, or 3 substituents independently selected from C₁-C₄ alkyl,C₁-C₄ alkoxy, and halo.
 4. The compound of claim 1, or a salt thereof,wherein A is a bicyclic heterocyclyl group that is unsubstituted.
 5. Thecompound of claim 1, or a salt thereof, wherein R¹ is C₈-C₁₄ alkyl. 6.The compound of claim 1, wherein the compound is selected from:

or a salt thereof.
 7. A composition comprising a compound claim 1 or asalt thereof, and a thermostable DNA polymerase.
 8. The composition ofclaim 7, wherein the thermostable DNA polymerase is selected from thegroup consisting of: Taq, Tca, Tfu, Tbr, Tth, Tih, Tfi, Tli, Tfl, Pfu,Pwo, KOD, Tma, Tne, Bst, Pho, Sac, Sso, ES4, or a mutant, variant orderivative thereof.
 9. The composition of claim 7, wherein the compoundis bound to the DNA polymerase and inhibits the activity of the DNApolymerase.
 10. The composition of claim 7, further comprising one ormore nucleic acid amplification reagents selected from the groupconsisting of: deoxynucleotide triphosphates, buffer, a magnesium salt,an oligonucleotide primer, and a nucleic acid template.
 11. Acomposition comprising a compound of claim 1 or a salt thereof, and oneor more nucleic acid amplification reagents selected from the groupconsisting of: a polymerase, deoxynucleotide triphosphates, buffer, amagnesium salt, an oligonucleotide primer, and a nucleic acid template.